Xiaomei Sun ^1,2,3 Mingyong Deng ^4* Chuxing Cheng ⁴ Ya Zhao ^1,2,3* Zuqin Liu ^1* Yu Yang ^4* Qiaoxia Xu ^4* Rong Yao ^4* Meilin Jin ^1,2,3,5,6* Chao Kang ^4,7*

• ¹ National Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, Hebei Province, China
• ² College of Animal Medicine, Huazhong Agricultural University, Wuhan 430070, China, Wuhan, Hebei Province, China
• ³ The Cooperative Innovation Center for Sustainable Pig Production, Huazhong Agricultural University, Wuhan, Hubei Province, China
• ⁴ Wuhan Keqian Biology Co., Ltd, Wuhan, Hubei Province, China
• ⁵ Hubei Province Animal Disease Comprehensive Prevention and Control Industry Technology Innovation Alliance, Wuhan, China
• ⁶ Hubei Jiangxia Laboratory, Wu han, China
• ⁷ Hubei Province Animal Disease Comprehensive Prevention and Control Industry Technology Innovation Alliance, Wuhan 430070, China, Wuhan, China

Mycoplasma synoviae (MS) is a globally important avian pathogen that can causes infectious synovitis, respiratory diseases in avian species, leading to serious economic losses in the poultry industry. Vaccination is one of the most cost-effective and efficient methods for preventing and controlling MS infections. Therefore, developing new and efficient vaccines is important in reducing the risk of MS infection. Despite significant advances in vaccine development, a commercially viable subunit vaccine against MS remains elusive. Here, we aim to address this gap by engineering a broad-spectrum, highly efficacious subunit vaccine. In this study, six clinical MS strains were isolated from different provinces in China. After whole-genome sequencing, we analyzed their common genes using Biopython software. We identified 22 genes that were shared among all strains; these genes had high copy numbers in highly virulent strains and, therefore, are potential antigen targets. Subsequently, by analyzing their properties, 10 candidate vaccine proteins were selected using Vaxign2 and IEDB Antibody Epitope Prediction. We then assessed the immune protective effects of every candidate protein and successfully developed a multivalent subunit vaccine composed of MSPB, Ppht, Cfba, and EF-G, which displayed the best protective effect among all the candidate vaccines tested. The immunization dosage of this combination vaccine was 20μg, with each protein accounting for 25%. The immune production period of the subunit vaccine is determined to be 28 days after the first immunization, with a duration of 180 days, and the immune protection rate against highly virulent strains reached 90～100%. These findings demonstrate that the combination of MSPB, Ppht, Cfba, and EF-G can be an efficient multi-component subunit vaccine, providing a new approach for developing vaccines against MS.